We measured this standard deviation, and it recapitulated the classification approach

ontrol cells.13 Reconciling this apparent paradox, when cells are exposed to irradiation, survivin relocates from the cytoplasm to the nucleus.13,14 Given that forced nuclear relocation eliminates cytoprotection, these data suggest that survivin may be posttranslationally modified when retained in the nucleus post-IR, indeed acetylation at K129, has been implicated in nuclear enrichment.15 During mitosis survivin is PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19837474 a chromosomal passenger protein, which operates in the chromosomal passenger complex with aurora-B, borealin and INCENP.The moral rights of the named author have been asserted. www.tandfonline.com Cell Cycle 261 Results and Discussion In this report whether the extreme C- and NH2-terminal ends of survivin are required for its ability to inhibit cell death, or for its essential function during mitosis was investigated. To do this 2 truncation mutants were generated, one in which the C-terminal 22 amino acids were removed, referred to as survivin1120; and the other lacking the first 10 amino acids, survivin11142. Expression constructs encoding these truncations were transfected into HeLa cells and cell lines stably expressing these forms were generated. GFP and survivin-GFP expressing cells were used as controls, see.19 Immunoblotting was used to verify the size of ectopic proteins and compare their levels of expression. Fluorescence imaging revealed that 90% of all cells were expressing these constructs, and like wild type both 262 Cell Cycle Volume 14 Issue 2 survivin1120 and survivin11142 were predominantly localized within the cytoplasm of interphase cells. Survivin terminal truncations can inhibit apoptosis To test whether these truncation mutants could protect cells against apoptosis, asynchronous cells expressing GFP, or the GFP tagged survivin forms indicated, were treated with TRAIL for 0, 60, or 90 minutes, whole cell lysates prepared, and apoptosis assessed using a caspase-3 tetrapeptide cleavage assay. In this assay lysates from cells overexpressing WT reduced caspase-3 activity compared with cells expressing GFP alone, as expected. Lysates prepared from cells expressing survivin1120 and survivin11142 were also able to protect cells against this apoptotic challenge, demonstrating that neither end of the protein is required to inhibit TRAIL- mediated apoptosis. NH2 truncation eliminates protection from X-irradiation Next to test the ability of survivin mutants to protect cells against a second death-signaling pathway, cells were exposed to increasing doses of X-rays. Survival was monitored using a clonogenic assay in which colonies of 50 cells or more were counted 7 d post-treatment, and plotted on a logarithmic scale as the “surviving fraction.” As shown in Localization of survivin truncations during mitosis To investigate whether the ends of survivin are required for cell division the localization of these truncation mutants during mitosis was examined. Survivin has a very distinct pattern of localization during mitosis.24, 25 It is centromeric until the metaphase-anaphase transition, after which it transfers to the central anaphase spindle, decorates the equatorial cortex at the site where the cell will form the cleavage furrow, and finally it is discarded from the cell during cytokinesis via midbody externalisation. Interestingly, although the C-terminal truncation, survivin1120 localized to the centromeres during early mitosis, it was not specifically BCTC price confined to these foci, instead it was distributed all along